Organophosphorus Compounds
The organophosphorus compounds (OPs) are highly toxic molecules which make up certain chemical warfare agents and pesticides. Some of these compounds such as paraoxon or parathion are used for their insecticidal property. In fact, they are easy to manufacture and are widely used for agriculture in developing countries. Unfortunately, this very widespread use is responsible for large numbers of cases of poisoning world-wide (200,000 deaths per year according to the WHO).
Most OPs are unstable products as they hydrolyze rapidly. They do not therefore persist in the environment in their toxic form. By contrast, certain products developed by armies are much more stable and dangerous, such as sarin, soman, tabun or VX. These chemical warfare agents are now of increasing interest to terrorists. Sarin in particular has already been used during attacks carried out by the Aum sect, in 1994 at Matsumoto and in 1995 in the Tokyo metro. Faced with these growing threats, the study and especially the development of effective means of decontamination has never been more urgent.
The organophosphorus compounds act by percutaneous absorption and by inhalation. They are very often colourless and odourless liquids. Poisoning with one of these agents rapidly becomes apparent (less than 1 minute to 60 minutes) due to characteristic and extremely serious symptoms (even the death of the poisoned subject). These molecules, once ingested into the human organism, have a neurotoxic effect. They attack an enzyme which is very important for the proper functioning of the nervous system: acetylcholinesterase. This enzyme is essential in the transmission of nerve messages. In fact, as the impulse passes from neuron to neuron, the electrical information is converted to a chemical message in the synaptic cleft. The molecules thus released are called neurotransmitter (for example: acetylcholine). Once released in the cleft, the acetylcholine mostly binds to the receptors of the post-synaptic neuron in order to ensure the continuity of the nerve message. The bound and non-bound molecules must then be re-trapped or degraded, thus allowing the regulation of the intensity and duration of the impulse. The role of the acetylcholinesterases is therefore to ensure that the nerve message stops, by degrading the acetylcholine in the synaptic cleft.
The OPs react rapidly with the serine at the active site of the acetylcholinesterases, forming an inactive phosphoenzyme. The covalent intermediate thus formed, the enzyme has lost all activity. These compounds therefore constitute irreversible inhibitors of these enzymes. The acetylcholine is then no longer degraded in the synaptic cleft and accumulates.
In order to be prepared for these dangers, prevention and decontamination protocols are provided. At present equipment is decontaminated using highly concentrated soda (NaOH). Protective suits and masks have been designed to prevent all contact with these agents. In case of the poisoning of humans, treatment with soda obviously cannot be envisaged. The victim is simply decontaminated using a solution of sodium hypochlorite (Javel water) and washed with copious amounts of soap and water. Foulon gloves also allow the liquid to be absorbed by the victim's skin. For cases of inhalation (percutaneous or not percutaneous) of neurotoxic agents, there is pretreatment with pyrostigmine, which can be taken in cachet form. This molecule reversibly blocks the acetylcholinesterases and prevents the OPs from binding to them. The individual's life is thus saved. Moreover, an emergency treatment of symptoms also exists in the form of self-injection syringes containing atropine (anticholinergic), diazepam (anticonvulsant) and pralidoxime (reactivator of the inhibited acetylcholinesterases). The injection must however be given immediately after poisoning in order to be effective. This does not however prevent the appearance of incapacitating sequelae.
Although some progress in prophylaxia has been made with the abovementioned techniques in the last twenty years, the treatments for these poisonings and existing protection nevertheless remain unsatisfactory. Unfortunately, all the pharmacological leads explored seem to come to an impasse. However, the emergence of the concept of a “bioscavenger” has given rise to new hopes of a more effective armamentarium. In fact, the idea of using enzymes capable of trapping and/or degrading the OPs on the skin and in the blood before they reach their neuromuscular and central biological targets is particularly attractive.
Human ButyrylCholinesterase (BuChe) is an enzyme similar to acetylcholinesterase, the physiological role of which is not clearly established. Despite that, it represents great hope as it traps the organophosphorus compounds in the blood route before they reach their targets (Raveh et al., 1993). Furthermore, the natural enzyme injected into humans is particularly stable, with a half-life of 11 days. However, this natural scavenger is in much too low a quantity in the blood to protect us naturally from the dangers of the OPs. In fact it acts as a stoichiometric binder of the OPs: one enzyme can only neutralize a single molecule. A rapid calculation makes it possible to show that huge quantities of enzyme are needed in order to obtain an effective treatment. The resources to be used then seem disproportionate and would correspond to a dose of 200 mg of protein per injection and per soldier. However, for want of something better, BuChE represents a concrete plan in particular for the American army which, at the end of 2006, provided for a million doses to be made available for its soldiers. The production of the enzyme is ensured by genetic engineering thanks to transgenic goats. The need for such a quantity of protein is nevertheless very expensive, and despite the resources utilized, this enterprise constitutes a major technological challenge. A few variants of BuChE having an OP-hydrolase activity do exist but their catalysis is very slow in comparison with enzymes capable of hydrolyzing the OPs naturally.
Human paraoxonase (HPON1) is an OP-hydrolase which has numerous advantages. Its protective role against OP poisoning has been established in mice. Furthermore, its human origin should avoid multiple injections of the therapeutic protocols inducing an immune response. HPON1 is a plasma protein mainly associated with HDL. The three-dimensional structure of the natural enzyme has not been resolved, only the structure of a human-mouse-rat-rabbit chimera of PON1, (Harel et al., 2004). Nevertheless, this structure has not made it possible to obtain more active mutants. Furthermore, a pharmacological use is impossible in the immediate future. In fact, all attempts to obtain a large quantity of active human paraoxonase have failed for technical reasons.
Other promising OP-hydrolases have been isolated. These are enzymes of the family of the phosphotriesterases (PTEs). These enzymes constitute true catalytic scavengers discovered in soil bacteria: in particular Pseudomonas diminuta and flavobacterium sp. (Munnecke, 1976) for the opd gene, and Agrobacterium radiobacter for the opdA gene (Jackson et al., 2005). The PTEs are enzymes which are extremely promising for the development of a bioscavenger for neurotoxic agents. But there are also fundamental concerns about these enzymes: in fact, the biological implication(s) (s) of the latter remain completely unknown. Furthermore, the catalytic mechanism of these extremely effective enzymes is somewhat obscure.
The PTEs are the most active of the enzymes known to degrade the OPs. Studying these could make it possible to carry out treatments for therapeutic (cutaneous and opthalmological) decontaminations which would advantageously replace the only existing effective means which is soda. The latter obviously cannot be used on living beings. Moreover, the PTEs would also be effective for decontaminating soil polluted with pesticides. They could also be used for detecting OP pollution. Thus, there are projects which attempt to chemically bind these proteins to a support and detect any catalysis by various means such as the detection of electric signals or by spectrophotometry. Another major asset is that the PTEs are capable of hydrolyzing a broad spectrum of OPs, such as parathion, paraoxon, soman, sarin and the most toxic of all, VX.
The hypotheses relating to the origin of this OP-hydrolase activity in bacteria are multiple and controversial, even though it seems more likely that this activity results from a structural similarity of its natural substrate to these poisons. Moreover, the physiological role of these enzymes remains unknown (Aubert et al., 2004). Several genes exist which are known to encode for mesophilic PTEs. A first gene (opd) was simultaneously isolated from P. diminuta and Flavobacterium sp., and encodes a protein of 365 amino acids. This protein possesses a peptide signal of 29 residues allowing its addressing in the periplasmic space. Another known gene (opda), isolated from A. radiobacter (Jackson et al., 2005), encodes a protein of 362 amino acids possessing a peptide signal of 33 residues. These two proteins share 90% sequence identity. Whilst these mesophilic PTEs are very active vis-à-vis the OPs, they are however expensive to produce, and unstable.
Recently, a novel protein of this family was isolated and purified (Merone et al., 2005). This metalloenzyme of 35.5 kDa possesses 31% sequence identity with the PTEs of P. diminuta and was isolated from the archaeon Sulfolobus solfataricus. This organism lives in extreme conditions (87°-93° C. and pH 3.5-5). The latter confer exceptional thermostability properties upon this protein. This is a hyperthermophilic enzyme the maximum activity of which occurs at approximately 95° C., and it is clearly less active vis-à-vis paraoxon than the PTEs of P. diminuta. Another hyperthermophilic PTE has been isolated from Sulfolobus acidocaldarius (Porzio et al., 2007). The hyperthermophilic PTEs are less active vis-à-vis the OPs than the mesophilic PTEs, but on the other hand have the advantage of being very stable and inexpensive to produce.
Bacterial Infections
Bacterial infections constitute one of the major causes of human pathologies. Some of these infections can be contracted in hospital and constitute a major public health problem. In France, according to the different studies carried out, approximately 5 to 10% of hospitalized individuals fall victim during their stay in hospital, i.e. 600,000 to 1,000,000 patients per year. On top of the pathologies initially responsible for the hospitalization, these infections aggravate the patients' vital prognosis (approximately 6000 deaths per year, the tenth cause of deaths in France). Besides this fact there is also the additional financial cost of prolonged stays in hospital and the provision of expensive treatment. These problems are further exacerbated by the appearance of a growing number of cases of antibiotic resistance. A certain number of strategies are being developed in order to acquire new tools against this resistance. One of the most promising leads involves disturbing communications between bacteria. In fact, although bacteria are single-cell organisms, they are capable of communicating with each other and thus responding collectively to an environmental change. These communication mechanisms, known as “quorum sensing” (QS), allow the synchronization and modulations of the expression of certain genes (Federle and Bassler, 2003; Fuqua and Greenberg, 2002; Whitehead et al., 2001). This communication is modulated by small “signal” molecules, capable of freely diffusing through the cell membranes and regulating the expression profiles of genes. Moreover, the QS phenomenon is not limited to the prokaryotes, since certain single-cell eukaryotic pathogens of algae also use QS for coordinating certain biological functions, such as virulence (Oh et al., 2001).
Of all the signals used for QS, the acyl homoserine lactones (AHLs) appear to be the most widespread (in particular in Gram-negative bacteria) and are the most studied.
Their involvement is demonstrated in numerous significant biological functions, such as symbiosis, conjugation, production of antibiotics, sporulation, virulence and biofilm formation (Fuqua and Greenberg, 2002; Whitehead et al., 2001; Zhang, 2003).
The concentration of these “signal” molecules is very significant and regulated in part by enzymes capable of degrading these compounds. In particular there are AHL acylases and AHL lactonases which are capable of degrading these lactones, such as AiiA, originating from Bacillus thuringiensis (Dong et al., 2002). In order to combat bacterial infections, the idea of disturbing quorum sensing is an extremely promising lead (Rasmussen and Givskov, 2006). In fact, given that QS mutant pathogens no longer express virulence genes and become non-virulent (Passador et al., 1993; Pirhonen et al., 1993), it therefore seems possible to control bacterial infections by attenuating the QS of pathogens.
Thus, the expression of a QS-attenuating enzyme: a “quorum quenching” (QQ) enzyme, whether this is an AHL lactonase or an AHL acylase, in plant or human pathogens such as Erwinia carotovora and Pseudomonas aeruginosa, significantly reduces their virulence (Dong et al., 2000; Lin et al., 2003; Reimmann et al., 2002). Furthermore, transgenic plants expressing a QQ lactonase are effectively resistant to pathogen infections (Dong et al., 2001).
Recently, the protein SsoPox, originating from the hyperthermophilic archaeon Sulfolobus solfataricus has been cloned and characterized for its phosphotriesterase activity (Merone et al., 2005). This protein is hyperthermostable with a denaturation half-life of approximately 4 hours at 90 to 95 and 100° C., respectively. This allows very effective and low-cost purification of the recombinant protein by heating the cell lysates, and thus precipitating the host proteins (Escherichia coli). In 2006, it was shown that SsoPox possesses significant AHL lactonase activity (Afriat et al., 2006).